Cancer is the second leading cause of death in the world and is often untreatable. Protein-based therapeutics, such as immunotherapeutics, show promising results in the fight against cancer, resulting in their market share increasing every year. Unfortunately, most protein-based therapeutics suffer from fast degradation in the blood, making effective treatment expensive, causing more off-target effects (due to the high doses necessary) and often require repeated injections to stay within the correct therapeutic range. Encapsulation of these proteins inside nanocarriers are prompted to overcome these problems by enhancing targeted drug delivery and, thus, leading to a less frequent administration and lower required dose. However, most current protein encapsulation methods show very low loading capacities (LC). This leads to even more expensive treatment and might pose further risk for the patient caused by systemic toxicity against high concentrations of carrier material. We investigated and optimized protein nanoprecipitation as a method to obtain a high protein LC and encapsulation efficiency (EE) inside poly(lactic-co-glycolic acid) (PLGA) nanoparticles via a simple two-step process. In this work we used model proteins to investigate the influence of various parameters such as precipitation solvent, addition speed and protein concentration on the protein activity and precipitation yield. Moreover, we have also characterized both protein nanoprecipitation and PLGA EE. We demonstrate LC up to 10%, which is a significant advancement compared to classical emulsion based encapsulation techniques. Our work is a critical step towards high-loading encapsulation of immunotherapeutics.

Immunotherapy is considered to be one of the most promising cancer treatments. However, applications of some promising immunotherapeutic, such as anti-fibroblast growth factor-inducible 14 (anti-Fn14), are limited due to their low bioactivity. Anti-Fn14 antibodies mimic TWEAK, a member of the tumor necrosis factor superfamily TNFSF ligands, which stimulate Fn14 (TNFRSF) which results in the induction of necrotic and apoptotic cell death.

It has previously shown that the bioactivity of such antibodies, could be improved by their crosslinking, that is immobilization of multiple antibodies in close proximity. In this work, we have developed a method to increase the activity of the anti-Fn14 antibodies by physical cross-linking on gold nanoparticles. We have developed and optimized the method for the preparation of gold nanoparticles, their functionalization with poly-ethylene glycol (PEG) linkers, and grafting of the antibodies on the surface.

We showed that antibodies can be successfully attached to nanoparticles without affecting their activity. Most importantly, the bioactivity measured of the grafted antibodies was increased in comparison to non-grafted antibodies as verified by the triggering HT-1080 cell line to produce the inflammatory cytokine IL-8 which was characterized by ELISA assay.

Our results suggest that conjugation of monoclonal IgG1 antibody on the inorganic nanoparticles is a very promising technique to boost the efficacy of the immunotherapeutic.

FUNDING:

This project has received funding from the European Union´s Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie grant agreement No 813871.

REFERENCES:

1. Medler, J. et al. (2019) ‘TNFRSF receptor-specific antibody fusion proteins with targeting controlled FcγR-independent agonistic activity’, Cell Death and Disease. Springer US, 10(3). doi: 10.1038/s41419-019-1456-x.
2. Kelley, S. K. et al. (2001) Preclinical Studies to Predict the Disposition of Apo2L/Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand in Humans: Characterization of in Vivo Efficacy, Pharmacokinetics, and Safety. Available at: http://jpet.aspetjournals.org (Accessed: 19 September 2019).
3. Alerts, E. (2019) ‘Cachectin / tumor necrosis factor : production , distribution , and metabolic fate in vivo . Information about subscribing to The Journal of Immunology is online at : METABOLIC FATE IN VIVO ’’.
4. Winkles JA. The TWEAK-Fn14 cytokine-receptor axis: Discovery, biology and therapeutic targeting. Nat Rev Drug Discov. 2008;7(5):411–25.

Pioglitazone is an oral anti-hyperglycemic agent and it is used for the treatment of diabetes mellitus type 2. The anti-inflammatory activity has also been demonstrated in the literature. Pioglitazone belongs to Class II of Biopharmaceutical Classification System, i.e., low soluble and high permeable. Polymeric nanoparticles formulations play an important role in the improvement of the efficacy of ocular therapies. These systems are non-toxic and biodegradable, show appropriate physicochemical characteristics as well as prolonged release profile suitable for ocular delivery.

An accurate, sensitive, selective, reproducible and high throughput LC-MS/MS method was validated to quantitate pioglitazone in ocular swine tissues (cornea, sclera, lens, aqueous humour and vitreous humour). The chromatographic separation was achieved in 10 min on a Kinetex C18 column at 35°C, using a mobile phase composed of formic acid 0.1% in water and formic acid 0.1% in acetonitrile in gradient mode at a flow rate of 0.6 mL/min. The detection in the MRM mode for pioglitazone was 357.2/134.1 for quantitation (most sensitive), and 357.2/119.1 for confirmation. Linear response of pioglitazone was observed over the range of 5–100 ng/mL. Tissues were spiked with pioglitazone to obtain final extract levels in ng. The limit of quantitation was in 10 ng/ml in extract. The recovery of pioglitazone was in the range 70-120% % in all tissues and levels tested. The intra-day precision was < 3% and the inter-day precision was <7%. The obtained extracts demonstrated to be stable under various experimental conditions in all the studied matrices. The recovery of pioglitazone spiked at the levels of the validation study, in the form of a nanoparticle formulation of polylactic-co-glycolic acid-polyethyleneglycol (PLGA-PEG), was also found in the range 70-120 % with a precision <7%. This method can be applied to in vivo and ex vivo biodistribution studies related to the ocular administration of pioglitazone nanoparticles.

Albendazole (ABZ) and benznidazole (BZL) are drugs used in parasitic infections treatment and classified as class II drugs by the Biopharmaceutical Classification System (BCS) due to their low solubility, which limits their bioavailability. In this research, solid dispersion (SD) technology was used to enhance ABZ and BZL performance by increasing their dissolution rate and solubility. SDs were prepared by the fusion method, employing a triblock co-polymer, Poloxamer 407 (P407), as carrier to disperse 32 of BZL or 50% w/w of ABZ. Furthermore, physical mixtures (PM) of P407 and either ABZ or BZL were prepared in the same drug/polymer proportion, and then SDs and PMs were characterized. Dissolution tests of SDs, PMs and commercial formulations (CF) of ABZ and BZL were carried out and dissolution profiles were analyzed with the lumped mathematical model, which allowed obtaining different parameters of pharmaceutical relevance. The results indicated that SDs of ABZ presented an initial dissolution rate (IDR) 21-fold and 11-fold faster than PM and CF, respectively, while the IDR of BZL SD was 2.5-fold and 4.5-fold faster than CF of BZL, respectively. In the case of samples containing BZL, the time required to reach 80% dissolution of the drug (t_80%) was 4 (SD), 46 (PM), and 239 min (CF); while the dissolution efficiency (DE) values determined at 30 minutes were 85 (DS), 71 (MF) and 65% (FC). For the samples containing ABZ, t_80% was 2 (SD), value not reached (PM) and 40 min (CF); while the DE values determined at 30 minutes were 85 (SD), 36 (MF) and 65% (CF). The results showed that the SDs developed notably increased the dissolution rate, in consonance with the values obtained from the pharmaceutical parameters, which could lead to faster absorption and, consequently, increase the bioavailability of these drugs that are poorly soluble in water.

The aim of this study is to investigate the impact of various mixtures of crosslinked polystyrene (CPS) and yttrium-stabilized zirconia (YSZ) beads (CPS:YSZ=0:1–1:0 v:v) on the breakage kinetics and energy consumption during the wet stirred media milling (WSMM) of fenofibrate (FNB, BCS II drug). Twenty WSMM experiments were conducted with various mixtures at different stirrer speeds (3000, 4000 rpm) and bead loadings (0.35, 0.50) with a suspension of 10% FNB, 7.5% HPC-L, 0.05% SDS. Laser diffraction, viscometry, XRPD, and PLM imaging were used for characterization. A new modified microhydrodynamic model considering bead packing was used to interpret the roles of different beads. The results showed YSZ beads provided faster breakage, which was supported by the microhydrodynamic results. On the other hand, CPS beads required lower stirrer power. When both cycle time and specific energy consumption were considered for process optimality, YSZ–CPS bead mixtures performed better than YSZ or CPS alone and the optimal bead mixture was found to depend on the stirrer speed and the bead loading.

The increase in life expectancy is favoring the prevalence of musculoskeletal diseases that compromise the quality of life of millions of people all over the world. Moreover, some life styles promote the early apparition of degenerative diseases. The unmet clinical demand of tissue regeneration prompts the searching of novel strategies. Cyclodextrins (CDs) have an outstanding track record as versatile tools for drug formulation. Although still less explored, CDs are being found useful for a variety of purposes in the design of advanced scaffolds for regenerative medicine. CDs can simultaneously host therapeutic agents and structural components of the scaffolds serving as reversible tie-junctions. Versatile 2D nanostructured systems can be obtained by means of electrospinning. Soft 3D supramolecular structures can be prepared as syringeable materials to be administered using minimally invasive techniques, with the additional advantage of ensuring a full filling of the gap in the damage tissue. Additionally, CDs have recently been demonstrated suitable to prepare patient-personalized scaffolds by means of 3D printing, opening novel approaches for scaffold design using additive manufacture techniques. 2D and 3D CD-based scaffolds have shown advanced performances for various tissues regeneration, including bone and cartilage, combining cell growth support and sustained delivery of active substances, growth factors and even viral vectors for improved tissue repair and angiogenesis. This talk aims to provide an overview of the state-of-the-art of the use of CDs in bone regeneration. Although most information still refers to in vitro and preclinical studies, recent progresses in the field point out CDs as key components of improved scaffolds.

Poor solubility, erratic bioavailability and delivery challenges associated with gliclazide, an oral anti-hyperglycemic agent commonly prescribed in type 2 diabetes mellitus (T2DM) can be overcome by exploring electrospun nanofibers technology [1]. Employing emulsion electrospinning method with polyvinyl alcohol (PVA) alone and in combination with poly (D, L-lactide-co-glycolide) (PLGA), nanofibers were fabricated as carriers for delivery of gliclazide [2, 3]. In this study, different concentrations of PLGA at 0.05, 0.01 and 0.15 %w/v was added to PVA to achieve a modified drug release profile to meet the typical physiological needs of T2DM, such as a faster drug release at the time of meals followed by prolonged drug release profile over an extended period to maintain constant plasma glucose level, is highly desirable for better T2DM management [1,4]. The fabricated gliclazide nanofibers were characterised by solubility studies, in vitro drug release studies, drug release kinetic studies, scanning electron microscopy studies (SEM), differential scanning calorimetric (DSC) studies and fourier transform infrared (FTIR) spectroscopy ( FTIR) studies. The formulation (GLZNF2) with Drug: PVA: PLGA 0.01: 10: 0.05 % w/v ratio produced optimized gliclazide nanofibers. The optimized formulation of gliclazide loaded nanofibers was incorporated into an empty gelatin capsule for oral administration [5, 6]. The SEM image of optimized formulation GLZNF2 shows the cylindrical shape of fiber indicates gliclazide was incorporated homogeneously in the polymer with the average fiber diameter of 4.357µm± 0.83. The solubility and dissolution rate of gliclazide nanofibers were significantly improved compared to pure gliclazide. The findings emphasize gliclazide nanofibers produce a biphasic drug release profile, initial fast burst release, followed by prolonged drug release. Fabricated gliclazide fibers in oral dosage form have tremendous potential as drug carrier and alternative technology for the improvement of solubility, dissolution rate, reduction in the dosing frequency and better blood glucose control could be explored in T2DM management.

References :

1. Panda BP, Krishnamoorthy R, Bhattamisra SK, Shivashekaregowda NKH, Seng L Bin, Patnaik S. Fabrication of Second Generation Smarter PLGA Based Nanocrystal Carriers for Improvement of Drug Delivery and Therapeutic Efficacy of Gliclazide in Type-2 Diabetes Rat Model. Sci Rep. 2019;9(1):17331. doi:10.1038/s41598-019-53996-4
2. Vashisth P, Raghuwanshi N, Srivastava AK, Singh H, Nagar H, Pruthi V. Ofloxacin loaded gellan/PVA nanofibers - Synthesis, characterization and evaluation of their gastroretentive/mucoadhesive drug delivery potential. Mater Sci Eng C. 2017;71:611-619. doi:10.1016/j.msec.2016.10.051
3. Jahangiri A, Davaran S, Fayyazi B, Tanhaei A, Payab S, Adibkia K. Application of electrospraying as a one-step method for the fabrication of triamcinolone acetonide-PLGA nanofibers and nanobeads. Colloids Surfaces B Biointerfaces. 2014;123:219-224. doi:10.1016/j.colsurfb.2014.09.019
4. Javadzadeh Y, Ahadi F, Davaran S, Mohammadi G, Sabzevari A, Adibkia K. Preparation and physicochemical characterization of naproxen-PLGA nanoparticles. Colloids Surfaces B Biointerfaces. 2010;81(2):498-502. doi:10.1016/j.colsurfb.2010.07.047
5. Poller B, Strachan C, Broadbent R, Walker GF. A minitablet formulation made from electrospun nanofibers. Eur J Pharm Biopharm. 2017;114:213-220. doi:10.1016/j.ejpb.2017.01.022
6. Ghafoor B, Aleem A, Najabat Ali M, Mir M. Review of the fabrication techniques and applications of polymeric electrospun nanofibers for drug delivery systems. J Drug Deliv Sci Technol. 2018;48:82-87. doi:10.1016/j.jddst.2018.09.005

Efavirenz (EFV), a non-nucleoside reverse transcriptase inhibitor, is a first-line treatment for adult and pediatric human immunodeficiency virus type 1 infection (HIV-1) both in adults and in children. EFV belongs to class II of Biopharmaceutical Classification System (BCS); that is, it is poorly water-soluble and highly permeable. Approaches to improve efavirenz dissolution include manipulation of polymorphs, the use of superdisintegrants, solid dispersions, nano-sized polymeric micelles and complexation with two derivatives of β-cyclodextrin: RAMEB and HPβCD. Inclusion into cyclodextrins, besides increasing solubility, brings aditional advantages such as taste masking and protection against hydrolytical instability. These will be helpful in creating aqueous pediatric formulations, which are currently unavailable. In the present work, the interaction of efavirenz with beta- and gamma-cyclodextrins is investigated, revealing that only gamma-cyclodextrin has the adequate size to acommodate the bulky molecules of efavirenz. A complete solid-state characterization of the gamma-cyclodextrin complex with efavirenz is presented, as well as the dissolution profile of the complex.

Nanoparticles (NPs) offer noteworthy advantages in the treatment of several diseases by prompting, among other beneficial actions, site‐specific delivery of drugs. Ultra-small nanostructured lipid carriers (usNLC) are no exception. These correspond to a class of NPs composed of a blend of solid and liquid lipids, the latter usually in a higher proportion, which promote a less ordered solid lipid matrix, providing a higher drug loading capacity, drug release modulation, and improved stability in comparison with other lipid nanoparticles. Several manufacturing methods have been described for obtaining usNLC. However, an in-depth process understanding is mandatory for a comprehensive knowledge allowing NLC property control.

In the present work, the hot high-pressure homogenization method, characterized by an easy scaling-up, simplicity, and ease of handling, is used to develop highly concentrated, small-sized NLCs.

Critical process parameters (CPPs) and critical material attributes (CMAs) are assessed to address the reproducibility of the manufacturing procedure, consistency among batches, long-term stability of the formulation, drug loading capacity and drug release.

In order to acquire an enhanced understanding of this method, a multivariable analysis is herein applied to inspect how the physicochemical properties of the usNLC were influenced by the variation of CPPs. These include HPH-time, HPH-pressure, while CMAs, such as lipid concentration, are also taken into consideration. The results show that a high lipid concentration (15% w/w), with an intermediate pressure and a short time in HPH seem to be the crucial parameters for obtaining both a small particle size (<100nm) and a narrow size distribution (polydispersity index <0.2) in usNLC prepared by the hot-HPH method, without affecting zeta potential (>|30 mV|).

In recent years, our knowledge of the biological effects of cyclodextrins has grown significantly. Cellular actions of cyclodextrins originate in their ability to form complexes with lipophilic biomolecules. Cyclodextrins can target different types of molecules according to their size, for instance, alpha-cyclodextrins form complexes with phospholipids, while beta-cyclodextrins can bind cholesterol or prostaglandin E2. Due to their interactions with the main membrane constituents, cyclodextrins can affect the barrier function of biological barriers or influence the function of membrane proteins. Nevertheless, cyclodextrins can enter the cells by endocytosis and affect the intracellular cholesterol storage. Based on these findings, 2-hydroxypropyl-beta cyclodextrin (HPBCD) received the orphan designation for the treatment of Niemann-Pick disease, type C. The endocytosis of cyclodextrins works in different cell types and can be applied in the delivery of drugs into the cells. Tissue distribution and pharmacokinetics of cyclodextrins could be further characterized by imaging techniques. Radiolabeled HPBCD and randomly methylated beta-cyclodextrin (RAMEB) were used to study their in vivo behavior by positron emission tomography recently. Interestingly RAMEB accumulation was detected in prostaglandin E2 (PGE2) positive tumors. These findings can promote further research and application of cyclodextrins in inflammation and tumor diagnosis or targeting. The presentation aims to give an overview of the main biological effects of cyclodextrins and the recent results of this research field.